Enzymes with an Expanded Amino Acid Alphabet

结合计算设计和定向进化是创造非生物反应酶的有力方法。然而，可获得的化学物质的范围受到自然规范氨基酸字母表的限制，这些氨基酸包含有限的功能，并且对于许多生物催化转化不是最佳的。遗传密码扩展（GCE）允许将“化学编程”的非规范氨基酸（ncAAs）选择性地安装到酶活性位点，从而提供了在自然界中没有表现出来的有机催化机制的机会。一个有用的例子是硫脲，它是一类有价值的小分子有机催化剂，能够通过双氢键相互作用激活羰基化合物，促进广泛的化学转化。在该小组之前，pyrlyyl - trna合成酶/PyltRNACUA对已经成功地编码了几种赖氨酸衍生的硫脲，以响应重新分配的琥珀色停止密码子（UAG）。我们目前正在努力扩大可编码硫脲的范围，到目前为止已经成功地纳入了一种尿素类似物。有了这些翻译成分，我们将能够在计算设计的活性位点内探索一系列化学转化，最终通过定向进化优化这些酶。因此，这种方法结合了蛋白质催化剂的效率和选择性，以及小分子有机催化剂的广泛反应性。

Combining computational design and directed evolution is a powerful approach to create enzymes for non-biological reactions. However, the range of chemistries accessible is restricted by Nature's alphabet of canonical amino acids, which contain limited functionality and are not optimal for many biocatalytic transformations. Genetic code expansion (GCE) allows the site-selective installation of 'chemically programmed' non-canonical amino acids (ncAAs) into enzyme active sites, providing opportunities to access organocatalytic mechanisms not represented in Nature. A useful example are thioureas, which are a valuable class of small-molecule organocatalysts capable of activating carbonyl compounds through dual hydrogen-bonding interactions to promote a broad range of chemical transformations.Previously in the group, pyrrolysyl-tRNA synthetase/PyltRNACUA pairs have successfully been engineered to encode several lysine-derived thioureas in response to reassigned amber stop codons (UAG). We are currently working on expanding the scope of encodable thioureas and have so far successfully incorporated one urea analogue. With these translation components in hand, we will be able to explore a range of chemical transformations within computationally designed active sites, eventually optimising these enzymes via directed evolution. This approach thus combines the efficiency and selectivity of protein catalysts, with the broad range of reactivities accessible with small molecule organocatalysts.